We previously performed genome-wide linkage analyses for susceptibility to ozone (O3) in the inbred mouse and identified significant quantitative trait loci (QTLs) for lung inflammation and hyperpermebility responses on chromosomes 17 and 4, respectively. The chromosome 17 QTL includes the candidate gene Tnf which encodes the proinflammatory cytokine tumor necrosis factor alpha (TNF-a); a candidate gene in the chromosome 4 QTL is Tlr4 (toll like receptor 4) which has been implicated in innate immune response to endotoxin and respiratory syncytial virus in mouse and humans. Targeted disruption of TNF-a receptors in susceptible mice significantly attenuated O3-induced inflammation and thus indicated a functional role for Tnf in the model. Supportive evidence for Tlr4 as a candidate gene in O3 susceptibility was provided by the observation that O3-induced lung hyperpermeability and inflammation were significantly different between C3H/HeJ (resistant) and C3H/HeOuJ (susceptible) mice which differ only at a missense mutation in the third exon of the Tlr4 gene in the C3H/HeJ strain. To begin investigation of the mechanism(s) through which Tlr4 modulates O3 susceptibility we tested the role of nitric oxide synthase (NOS), which has previously been found to have an important role in Tlr4-mediated endotoxin responsiveness. Targeted disruption of the gene for inducible NOS (Nos2) significantly attenuated the permeability response to O3 in susceptible mice compared with normal wild type mice. Further, basal and O3-induced Nos2 gene expression, as well as NOS activity, were significantly diminished in Tlr4-mutant C3H/HeJ mice compared with wild type C3H/HeOuJ controls. These experiments are consistent with a role for NOS in O3-induced lung permeability, and suggest that Nos2 expression is mediated through Tlr4. Future studies will utilize global gene expression arrays in the C3H/HeJ and C3H/HeOuJ model to identify additional genes/pathways through which Tlr4 mediates differential susceptibility to O3-induced lung inflammation and injury.